1. Digital PCR outperforms quantitative real-time PCR for the detection and quantification of major periodontal pathobiontsHaris Munjaković, Katja Povšič, Mario Poljak, Katja Seme, Rok Gašperšič, Lucijan Skubic, 2025, izvirni znanstveni članek Povzetek: Background: This study comparatively evaluated the analytical and diagnostic performance of a multiplex digital polymerase-chain reaction (dPCR) assay and a quantitative real-time PCR (qPCR) for the simultaneous detection and quantification of periodontal pathobionts: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. Materials and methods: Subgingival plaque samples from 20 periodontitis patients and 20 periodontally healthy controls were analyzed. Several analytical parameters of the dPCR assay, optimized using DNA standards, were assessed versus qPCR: dynamic range linearity, precision, accuracy, prevalence, sensitivity, specificity, and concordance. The statistical analyses included Mann-Whitney U test, Wilcoxon test, McNemar's test, and Bland-Altman plots. Results: dPCR showed high linearity (R2 > 0.99) and lower intra-assay variability (median CV%: 4.5%) than qPCR (p = 0.020), with comparable accuracy and agreement. dPCR demonstrated superior sensitivity, detecting lower bacterial loads, particularly for P. gingivalis and A. actinomycetemcomitans. The Bland-Altman plots highlighted good agreement at medium/high loads but discrepancies at low concentrations (< 3 log10Geq/mL), resulting in qPCR false negatives and a 5-fold underestimation of the prevalence of A. actinomycetemcomitans in periodontitis patients. High concordance between the assays was observed for F. nucleatum across both study groups. Conclusions: dPCR outperformed qPCR for quantifying periodontal pathobionts and had superior sensitivity and precision, making it particularly effective in detecting low-level bacterial loads.
Ključne besede: digital PCR, oral microbiology, periodontal disease, quantitative real-time PCR, subgingival plaque
Objavljeno v DiRROS: 15.04.2026; Ogledov: 165; Prenosov: 106
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2. Development of a multi-targeted real-time PCR assay for the detection of the grapevine pathogen Xylophilus ampelinusAleksander Benčič, Alexandra Bogožalec Košir, Janja Matičič, Manca Pirc, Neža Turnšek, Tanja Dreo, 2025, izvirni znanstveni članek Povzetek: Background Xylophilus ampelinus is a plant pathogenic bacterium that causes bacterial blight in grapevines, which can lead to severe yield losses and economic damage. Owing to its fastidious growth on culture media, detection is primarily based on molecular methods. However, existing tests have produced inconsistent results, particularly when used to detect latent infections and non-validated matrices. There is a risk of false-positive results, with economic consequences such as restrictions on international trade. To enhance the diagnostics of X. ampelinus, a genome-informed approach was utilised to identify new potential targets for specific detection. On the basis of these sequences, multiple real-time PCR assays were designed, and their specificity and sensitivity were assessed, as well as their performance validated across three different grapevine tissues, including leaves, roots, and xylem. Results The newly designed real-time PCR assays were evaluated via high throughput testing for specificity and sensitivity and compared with a reference assay. The most promising assays were selected and validated in different grapevine tissues and included in a test performance study to validate their reproducibility and robustness. Three new assays (Xamp_BA_2, TXmp22.4, and Xamp_BA_7) demonstrated high specificity and sensitivity for X. ampelinus detection. The Xamp_BA_2 assay exhibited the best overall performance, offering high diagnostic sensitivity and robustness across diverse plant matrices. Importantly, the assays exhibited no cross-reactivity with non-target bacterial species and maintained high detection accuracy across diverse grapevine tissue types. Conclusions The newly developed real-time PCR assays provide an enhanced diagnostic framework for the detection of X. ampelinus in various plant matrices, significantly improving the applicability of molecular testing. The Xamp_BA_2 assay demonstrates superior performance and is recommended for routine diagnostics, with other validated assays being employed for confirmation of identification. The development of these new assays represents a significant expansion of our toolkit for the precise detection of X. ampelinus in grapevines, with the potential to contribute to the mitigation of grapevine bacterial blight, the prevention of yield losses, and the protection of international trade in grapevine material. Further implementation of these assays will support regulatory and phytosanitary efforts to mitigate the spread of X. ampelinus.
Ključne besede: Xylophilus ampelinus, grapevine bacterial blight, molecular diagnostics, Vitis vinifera, real-time PCR, genome-informed assay development
Objavljeno v DiRROS: 05.09.2025; Ogledov: 756; Prenosov: 390
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3. Test performance study on qPCR assays for detection of Phyllosticta citricarpaTjaša Jakomin, Janja Zajc Žunič, Polona Kogovšek, 2025, izvirni znanstveni članek Povzetek: Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, significantly affects citrus fruit marketability and can lead to premature fruit drop. Accurate and reliable detection of this quarantine pathogen is crucial, particularly for asymptomatic plant material. This study evaluated two qPCR assays, the EPPO recommended assay PC and assay Pc-TEF1, based on TEF region, for detecting P. citricarpa through a collaborative test performance study (TPS). DNA from the isolates of Phyllosticta spp. and other fungi was spiked into citrus fruit peel extracts (lemon, orange, and pomelo) and distributed among 13 laboratories. Sample and qPCR assay stability under typical transport conditions was confirmed, although prolonged storage affected Pc-TEF1 assay performance. The assays were assessed based on sensitivity, specificity, reproducibility, and repeatability. Both assays demonstrated high performance, with repeatability and reproducibility exceeding 95%. The PC assay, as expected, detected different related Phyllosticta species, while Pc-TEF1 showed higher specificity for P. citricarpa included in the TPS alone. Additionally, inhibitory effects were observed specifically in the pomelo peel samples, suggesting matrix-dependent variability. This TPS confirms that both PC and Pc-TEF1 qPCR assays are robust. Further evaluation of the qPCR assays would support the selection of the most reliable assays for the detection of P. citricarpa, contributing to the effective management of CBS disease in citrus production and trade.
Ključne besede: test performance study, Phyllosticta citricarpa, real time PCR, TEF1, biotechnology
Objavljeno v DiRROS: 07.05.2025; Ogledov: 1001; Prenosov: 827
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4. One-step RT-droplet digital PCR : a breakthrough in the quantification of waterborne RNA virusesNejc Rački, Dany Morisset, Ion Gutiérrez-Aguirre, Maja Ravnikar, 2014, izvirni znanstveni članek Povzetek: Water contamination by viruses has an increasing worldwide impact on human health, and has led to requirements for accurate and quantitative molecular tools. Here, we report the first one-step reverse-transcription droplet digital PCR-based absolute quantification of a RNA virus (rotavirus) in different types of surface water samples. This quantification method proved to be more precise and more tolerant to inhibitory substances than the benchmarking reverse-transcription real-time PCR (RT-qPCR), and needs no standard curve. This new tool is fully amenable for the quantification of viruses in the particularly low concentrations usually found in water samples.
Ključne besede: waterborne virus, quantification, droplet digital PCR, real-time PCR, digital PCR
Objavljeno v DiRROS: 04.03.2025; Ogledov: 1097; Prenosov: 716
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5. Grapevine flavescence dorée (FD) follow up Vitisens, GRAFDEPI and Qdetect (GRAFDEPI2) : final reportMarina Dermastia, Helga Reisenzein, Luca Ferretti, 2015, končno poročilo o rezultatih raziskav Povzetek: Europe is the world’s main producer and exporter of grapevine planting material and wine. This important economic sector is facing epidemic threats of at least 10 grapevine yellows diseases (GY) caused by phytoplasmas. In Europe the main phytoplasmas associated with GY are ‘Candidatus’ phytoplasma solani’ (BNp), which is a causal agent of bois noir and FDp, which causes flavescence dorée. Phytoplasmas are notoriously difficult to detect and identify and their specific detection relies exclusively on the molecular methods. Recently new methods, which are reliable, sensitive, fast, less expensive and suitable for using onsites, have been introduced. Among them is a loop-mediated isothermal amplification (LAMP) method, with several advantages (e.g. low sensitivity to plant extracts inhibitors, speed, robustness, simplicity of use) over the other methods (e.g. the real-time PCR). In a recently finished FP7 project VITISENS, a new LAMP protocols have been developed for specific detection of FDp, however, they have not been tested in the interlaboratories trials. In addition, there is no validated LAMP protocol available for the specific detection of BNp at the moment.
The main objectives of this project were:
(1) Development of new loop-mediated isothermal amplification (LAMP) based protocols for accurate, reliable, fast and affordable diagnostics of ‘Candidatus Phytoplasma solani’ (BNp), which will be applicable in-field
(2) To study new possible hosts plants and insect vectors of phytoplasma FDp.
(3) To organize an interlaboratory test performance study (TPS) to obtain validation parameters for the selected LAMP protocols for BNp, as well as for the LAMP assay for FDp detection developed in the course of the FP7 project VITISENS.
Ključne besede: phytoplasmas, grapevine yellows diseases, LAMP, real-time PCR
Objavljeno v DiRROS: 16.09.2024; Ogledov: 1168; Prenosov: 612
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9. Development of LAMP based protocol for accurate, reliable, fast and affordable diagnostics of Candidatus Phytoplasma solani : Euphresco success stroryMarina Dermastia, elaborat, predštudija, študija Povzetek: Phytoplasmas are cell-wall-free plant pathogenic bacteria; they have a broad range of plant hosts and diseases of many important crops are associated with these pathogens. At least ten phytoplasma ribosomal subgroups have been associated with grapevine yellows diseases, which have great economic impact on viticulture. In Europe, the main phytoplasmas associated with grapevine yellows are the causal agent of flavescence dorée and ‘Candidatus Phytoplasma solani’, which cause bois noir.
Ključne besede: phytoplasmas, grapevine yellows diseases, LAMP, real-time PCR
Objavljeno v DiRROS: 03.09.2024; Ogledov: 1275; Prenosov: 799
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10. Retrospective survey of Dickeya fangzhongdai using a novel validated real-time PCR assayŠpela Alič, Katarina Bačnik, Tanja Dreo, 2024, izvirni znanstveni članek Povzetek: Dickeya fangzhongdai, an aggressive plant pathogen, causes symptoms on a variety of crops and ornamental plants including bleeding canker of Asian pear trees. Historical findings stress the need for a specific detection tool for D. fangzhongdai to prevent overlooking the pathogen or assigning it to general Dickeya spp. Therefore, a qualitative real-time PCR for specific detection of D. fangzhongdai has been developed and validated. The developed assay shows selectivity of 100%, diagnostic sensitivity of 76% and limit of detection with 95% confidence interval in plant matrices ranging from 311 to 2,275 cells/mL of plant extracts. The assay was successfully used in a retrospective survey of selected host plants of relevance to Europe and environmental niches relevant to D. fangzhongdai. Samples of potato tubers and plants, plants from the Malinae subtribe (apple, pear, quince, and Asian pear tree) and fresh surface water from Slovenia were analyzed. D. fangzhongdai was not detected in any plant samples, however, 12% of surface water samples were found to be positive.
Ključne besede: molecular testing, diagnostics, plant pathogen, real-time PCR, Dickeya, survey, water
Objavljeno v DiRROS: 07.08.2024; Ogledov: 1360; Prenosov: 869
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